Resorptive sheet material for closing and healing wounds and method of making the same

ABSTRACT

A resorptive sheet material for closing and healing wounds substantially consists of a glycoprotein matrix containing substances which cause and, respectively, are conductive to the coagulation of blood including fibrinogen and thrombin. The sheet material is dry and of multi-layered structure, wherein at least one layer is free from thrombin and contains in the glycoprotein matrix thereof the fibrinogen in a substantially homogeneously distributed form, and at least one further layer is free from fibrinogen and contains in the glycoprotein matrix thereof the thrombin in a substantially homogenenously distributed form. The glycoproteins coming into consideration, above all, are non-linked fibrin, fibrin fission products, collagen, globulin, myoglobulin, casein and/or albumin. 
     Preferably, at least one of the layers, especially one or both outer layer(s), contains fibroblast cells present in dry state and/or agents conducive to the growth of the fibroblasts and their spreading-in, especially chondroitin sulphate. 
     For the production, fibrinogen is added to a thrombin-free, predominantly aqueous solution and/or suspension of the glycoprotein and said solution and/or suspension is processed to form a sheet-material layer, and onto the full surface of this layer at least one further layer is applied which, in its turn, has been obtained from a further, fibrinogen-free and thrombin-containing solution and/or suspension of the same or of another glycoprotein. In particular, a solution and/or suspension of one composition is applied onto an inert surface, where it is subjected to deep-freezing, and onto the resultant ice-layer surface, when still in the deep-frozen state, a solution and/or suspension of different composition is applied and subjected to deep-freezing, and, finally, all layers are jointly subjected to lyophilization.

This a divisional of application Ser. No. 486,580, filed on Apr. 19,1983, now U.S. Pat. No. 4,606,337, patented Aug. 19, 1986.

The invention relates to a resorptive sheet material for closing andhealing wounds, substantially consisting of a glycoprotein matrixcontaining substances which cause and, respectively, are conducive tothe coagulation of blood including fibrinogen and thrombin.

A sheet material for healing wounds has already been known the matrix ofwhich may consist of gelatin, collagen, polyglycolic acid or polylacticacid; this matrix material has the blood coagulation factor XIII andthrombin fixed thereto (DE-OS No. 29 14 822). This known sheet materialdoes not contain fibrinogen.

Furthermore, a tissue adhesive, called "fibrin adhesive", has been known(cf. DE-OS No. 30 02 933) which mainly consists of a deep-frozenfibrinogen solution with additions of factor XIII and a fibrinolysisinhibitor, such as e.g. aprotinin. For practical use, the deep-frozenfibrinogen solution is thawed, a solution of thrombin and calciumchloride is added thereto, the mixture is kept for some time until thestarting polymerization reaction becomes noticeable by an increase ofviscosity and then this reacting mixture is applied onto the tissueparts which are to be joined. In many cases, the expenditure forpreparing a tissue adhesive ready for use and the low lifetime of thepreparation which is ready for use have proven to be disadvantageous.

Further, it has already been proposed (Wiener Medizinische Wochenschrift7", pp. 86-89 (1976)) to heat lyophilized fibrinogen to a temperature of37° C., to apply the resultant solution onto a collagen fleece and tocause it to coagulate thereon by adding an aqueous solution of thrombinand an aqueous solution of factor XIII. Thereupon, the reactive sheetmaterial is transferred onto the wound. In this case, too, themanipulation causes difficulties since the sheet material which is readyfor use must be prepared directly beforehand and the short-time intervalof the preparation ready for use cannot reliably be determined.

As in an aqueous medium, and even already in moist surroundings,thrombin causes the polymerization and linking of fibrinogen intofibrin, preparations which contain both thrombin and fibrinogen mostlysuffer from poor storage stability.

Further, it is desirable that the sheet materials of the kind concernedcontain an agent which is intended to assist in the healing of thewound.

Based thereon, it is the object of the present invention to provide asheet material for closing and healing wounds which comprises thrombinand fibrinogen, which may be stored at ambient temperatures forprolonged periods of time without any appreciable loss of activity, andwhich is applied directly onto the wound, i.e. without adding further,e.g. activating components, where it is completely resorptive. Accordingto a further objective of the invention the sheet material shalladditionally contain one or more agents specifically assisting in thehealing of wounds.

Furthermore, this invention provides at least a method of making such asheet material which can be carried out easily, which in the course ofproduction does not affect the biological activity of fibrinogen andthrombin and, where applicable, of the agent(s) specifically assistingin the healing of wounds, and which permits a wide range of variation asregards the kind, number, concentration and arrangement of thecomponents within the sheet material.

Based on a resorptive sheet material for closing and healing wounds,substantially consisting of a glycoprotein matrix and containingsubstances which cause and, respectively, are conducive to thecoagulation of blood including fibrinogen and thrombin, the solution ofthe above object, which is provided by the present invention, ischaracterized in that the sheet material is dry and of multi-layeredstructure, at least one layer of the multi-layered sheet material isfree from thrombin and contains in the glycoprotein matrix thereof thefibrinogen in a substantially homogeneously distributed form; and atleast one further layer of the multi-layered sheet material is free fromfibrinogen and contains in the glycoprotein matrix thereof the thrombinin a substantially homogeneously distributed form. According to afurther preferred aspect of the invention at least one layer--preferablyat least one of the outer layers--of the sheet material additionallycontains fibroblast cells which are present in dry state.

According to a further preferred aspect of the invention at least onelayer--preferably at least one of the outer layers containing thrombinand fibroblast cells--of the sheet material additionally contains one ormore agents conducive to the growth and the spreading-in of fibroblastcells.

The method according to the invention for making such a sheet materialprovides adding fibrinogen to a thrombin-free, predominantly aqueoussolution and/or suspension of the glycoprotein and processing saidsolution and/or suspension to form a sheet-material layer, addingthrombin to a further, predominantly aqueous fibrinogen-free solutionand/or suspension of the same or of another glycoprotein and processingsaid solution and/or suspension to form a further sheet-material layer,and applying said layers on each other over the full surface thereof, ifdesired, including further, similarly prepared layers. Preferably,active fibroblast cells and/or an agent conducive to the growth as wellas the spreading-in of fibroblasts is added to the thrombin containingsolution and/or suspension of the glycoprotein. Preferably, chondroitinsulphate is used as the agent which is conducive to the growth and thespreading-in of fibroblasts.

A particularly preferred embodiment of this method provides applying asolution and/or suspension of one composition of the mentioned kind ontoan inert surface, where it is subjected to deep-freezing, and alsodeep-freezing on the resultant ice-layer surface, when still in thedeep-frozen state, a further solution and/or suspension of differentcomposition and, finally, jointly subjecting all layers tolyophilization. If desired, the applied solution and/or suspension maybe foamed prior to the deepfreezing operation.

Advantageous further embodiments and modifications of the invention areapparent from the subclaims.

The invention provides a completely resorptive sheet material forclosing and healing wounds, which may be stored under sterile, dryconditions at room temperature for prolonged periods of time, e.g. fortwo years and longer, without any appreciable loss of activity andwhich, after removal from its sterile package, is applied directly ontothe wound and stops the haemorrhage within a short time, e.g. withinapprox. 2 min., due to the increased offer of biologically activethrombin and fibrinogen.

The multi-layered embodiment of the sheet material permits a spatialseparation of fibrinogen and thrombin and an enrichment of componentsconducive to the wound healing, here especially the fibroblast growth,in at least one of the surface layers of the sheet material. This is whythe storage stability is so excellent without any appreciable decreaseof the biological activity. Further, the requirements as to theexclusion of moisture are not so high.

Besides, it is possible due to the multi-layered structure respectivelyto provide for the thrombin and the fibrinogen a particularly adaptedenvironment. The thrombin is contained in that outer layer which isapplied directly onto the wound, the so-called contact layer. Therefore,the thrombin shall be distributed in a matrix which may be rapidlysoaked and which is highly stable under mechanical load in order tosupport the further layers. The matrix materials coming intoconsideration for the thrombin-containing layer preferably are plasmaproteins (albumins, globulins) or fibrin fission products. Thefibrinogen, on the other hand, shall be distributed in a matrix whichmay be incorporated into the developing fibrin network and may berapidly decomposed physiologically. Therefore, the matrix materialspreferably provided for the fibrinogen-containing layer areglycoproteins, such as collagen, non-linked fibrin or fibrinpolypeptides. However, mostly such substances have a lower stabilityunder mechanical load, e.g. a lower tear resistance or tensile strength.Therefore, according to another aspect of the invention differentglycoproteins which are particularly matched to the respectiverequirements are provided for the various layers.

Due to the spatial separation, the common application of fibrinogen andthrombin on the support material is not necessary. Both fibrinogen andthrombin dissolve readily in water or predominantly aqueous solutionswith further water-miscible solvents. The application of an aqueoussolution containing both fibrinogen and thrombin is out of the questionsince fibrin would form from the fibrinogen and would polymerize.

However, when using a suspension of fibrinogen and thrombin in anorganic solvent, there is a risk of a partial denaturing with theconsequence of a loss of activity of the coagulation enzymes.

The specified multi-layered structure permits observance of preciselydefined fibrinogen and thrombin quantities as well as of predeterminednumbers of fibroblast cells, of which at least a fraction may bereactivated, per unit of area and/or unit of volume of the sheetmaterial. Preferably, the fibrinogen or thrombin and, where applicable,the fibroblast cells and/or the agent conducive to the spreading-in offibroblasts is dissolved and, respectively, distributed directly in thepredominantly aqueous solution and/or suspension of the glycoprotein,which is used for the preparation of the respective layer, so that inthe finished sheet material a substantially homogeneous distribution offibrinogen and, respectively, thrombin and the further components in therespective layers is ensured. This distribution ensures a rapid and highefficiency, which cannot be achieved to the same degree e.g. in theapplication of a crystalline, powdery mixture of fibrinogen and thrombinon a support material.

Further, the multi-layered structure permits the application of reactiveintermediate layers without any particular intrinsic mechanicalstability because the outer layers ensure the stability, in particularthe tensile strength of the sheet material.

Finally, the multi-layered structure ensures a wide range of variationas regards the adjustment of the kind, number and/or concentration ofthe various components in the layers to the requirements.

The method according to the invention can be carried out most easily,manages with predominantly aqueous solutions and/or suspensions and thusprevents any loss of activity of fibrinogen and thrombin and activefibroblast cells in the production.

The essential components of the sheet material according to theinvention have been known and may be prepared and produced according toknown methods and/or obtained in specialized trade.

The sheet material consists mainly of glycoprotein of which thesheet-material layers are composed and which serves as a supportmaterial for the coagulation enzymes. The glycoprotein is to becompletely resorptive and biologically decomposable since it isincorporated into the fibrin network finally covering the wound.Preferably such glycoproteins are provided as have a certain haemostaticeffect already on account of their chemical nature and/or the nature oftheir surface, as described e.g. in respect of collagen. Animal collagenis particularly suited due to its non-antigenic properties. However, allother glycoproteins must be obtained from human material. When moistenedwith the exudation of a wound, the support material shall absorb theliquid, dissolve partially and form a highly viscous, sticky paste,which adheres to the wound area, withstands the pressure of the issuingblood and activates the coagulation enzymes of the contacting blood.

Within the scope of the invention, preferably non-linked fibrin, fibrinfission products, collagen, globulin, myoglobulin, casein or albumincome into consideration as glycoprotein. In certain cases, a layer isalso composed of two or more of these glycoproteins.

Non-linked fibrin may be prepared from factor XIII-free fibrinogen andthrombin. An equal product is obtained in the reaction of the fibrinogenwith calcium-free thrombin and cysteine for blocking the SH groups ofthe factor XIII. The resultant fibrin is then non-linked, may, withoutfiber formation, be lyophilized and pulverized and may in this form beadded to a collagen solution.

Fibrin fission products are prepared by the tryptic decomposition ofhuman fibrin. After proteolysis, the material is pulverized and may beused in this form, whereby a highly water-soluble preparation isobtained.

An aqueous solution of collagen of animal origin may be obtained inspecialized trade, e.g. from the company Pentapharm, Basel, Switzerland.

Suitable globulin is precipitated at a 50% ammonium sulphateconcentration (i.e. one part of plasma per one part of aqueous,saturated ammonium sulphate solution) with human plasma. The product iscentrifuged off, dialyzed and lyophilized.

The further suited glycoproteins, such as myoglobulin, casein andalbumin, are known substances and also available in specialized trade.

What is particularly preferred as glycoproteins are collagen,collagen-like materials and fibrin fission products, which preferablyform the skeleton material for the fibrinogen-containing layer, as wellas non-linked fibrin and fibrin polypeptides, which preferably form theskeleton material for the thrombin-containing layer.

The layer may in different manners be composed of the glycoprotein.After the conventional preparation, various of the mentionedglycoproteins are obtained already in a leaf-like structure, asdescribed particularly in respect of collagen and modified collagens.Such leaf structures may serve as a layer of the sheet materialaccording to the invention after impregnation with an aqueous solutionof fibrinogen or thrombin. Another possibility is to spin theglycoprotein and to produce a fleece from the resultant fibers in thewet or dry process, which fleece is then impregnated with the aqueoussolution of fibrinogen or thrombin. Furthermore, the layer may have aporous foam structure. For this purpose, the glycoprotein issubstantially dissolved in a predominantly aqueous solution. It is notnecessary to produce a true homogeneous solution; rather, theglycoprotein may be processed also to form gel-like or gelatinouscompositions. Moreover, powdery or other fine-particle glycoproteins maybe present suspended and/or swelled in aqueous solution. As the solventthere is mainly used water or a solvent composition, which, apart fromlow amounts of water-miscible organic solvents, such as dioxan, lowerglycols, ethanol, mainly consists of water.

An aqueous solution of the respective coagulation protein is added tosuch a predominantly aqueous solution and/or suspension of theglycoprotein, thoroughly intermixed and the mixture is then deep-frozenin an inert mold as a thin layer and lyophilized. Before thedeep-freezing operation, the mixture may be foamed by means of inertfoaming agents (N₂,CO₂), wherein a certain pore size may be adjusted byadding surface-active agents. A foam structure with a large surface areais obtained for the layer(s). If all layers are foamed, there willresult an interengagement of adjacent layer surfaces, whereby the bondand the stability of the sheet material are increased. Preferably, thedesired number of layers is built up in successive steps, and thefinished layer composition is lyophilized. The product obtainedthereafter may readily be pulled off the mold surface, is flexible andmanipulatable without any special cautionary measures and may easily becut, packaged and sterilized. In order to prevent a premature activationof the coagulation enzymes, the final treatment steps are to be carriedout under exclusion of moisture so that a dry sheet material will beobtained and packaged moisture-proof. If required, a known drying agent,such as e.g. silica gel, may be provided within the sheet-materialpackage and fixedly provided on the latter to keep the sheet materialdry even for prolonged periods of storage.

Preferably, the sheet material according to the invention is intendedfor use with human beings. Therefore, the used fibrinogen preferably hasbeen obtained from human plasma. Suitable preparations are commerciallyavailable and may be obtained e.g. from the company Bering-Werke,Marburg. Furthermore, a preparation which is well suited may be isolatedaccording to the following process.

Human plasma is cooled to 4° C. and β-alanine (2 molar solution inethanol) is added thereto with agitation until with further ethanoladdition the raw fibrinogen precipitates. This raw fibrinogen iscentrifuged off, dissolved in 0.01M of tris buffer (pH 7.4) and againprecipitated by adding 2M of glycine. The isolated sediment is dissolvedin an 0.9% aqueous NaCl solution, dialyzed relative to the same solvent,desalted and subsequently lyophilized. The resultant microcrystallinefibrinogen has a molecular weight of 340,000±5%, is slightly digestedpartially in the α-chain, quickly dissolves after introduction into bodyfluid and immediately thereupon, e.g. within less than 2 min., starts topolymerize. The proportion of fibrinogen which is coagulatable insolution amounts to at least 85%. 10 parts by weight of such fibrinogencontain less than 0.1 parts by weight of cryo-insoluble globulin. It hasbeen found that the less the amount of cryo-insoluble globulin, the morerapid the fibrin polymerization. Therefore, such a fibrinogen depletedin cryo-insoluble globulin is used preferably.

The fibrinogen content of the fibrinogen-containing layer may range from0.1 to 30 mg, preferably from 0.5 to 10 mg per 1 cm³ of glycoproteinmatrix.

The thrombin provided as a further necessary coagulation enzyme servesas an initiation substance for the fibrin formation and shortens thereaction time of the fibrinogen conversion in the issuing blood. Underknown, standardized conditions the thrombin shall at least have abiological activity of 10,000 international units/mg of thrombin.Suitable preparations are commercially available. E.g. a suitablethrombin in microcrystalline form having a biological activity of atleast 3,000 units/mg of the preparation (which apart from thrombincomprises known stabilizers and support materials) may be obtained underthe tradename "Topostasin" from Hoffmann LaRoche, Grenzach, Baden.Furthermore, part of the thrombin may be replaced by prothrombin.Prothrombin constitutes a stable thrombin store, which may be shelvedfor long periods of time and, in the case of access of moisture, isactivitated by thrombin being present and/or the issuing blood.Prothrombin is sold e.g. as a PPSB preparation by the company Imuno-AG,Vienna. A combined preparation containing thrombin and prothrombin maye.g. be separated from a commercially available prothrombin complex bycolumn chromatography or be extracted from human plasma by means ofbarium sulphate and be recovered from the crystalline precipitate.

The thrombin content including the thrombin available from prothrombinshall range from 10 to 2,000 units preferably from 50 to 1,000 units per1 cm³ of the glycoprotein matrix. These units correspond to theinternationally common NIH units (National Institute of Health).

Apart from the coagulation enzymes fibrinogen and thrombin, the sheetmaterial according to the invention may comprise further substancesinfluencing the coagulation of the blood. These include e.g.fibrinolysis inhibitors which prevent the redissolution of the alreadyformed fibrin clot. Suitable fibrinolysis inhibitors are e.g.antiplasmins, such as aprotinin, α₂ -antiplasmin, α₂ -macroglobulinand/or trypsin inhibitor. Furthermore, phospholipids, obtained e.g. fromcerebral matter or a thrombocyte preparation, may be added. Besides, forthe specific treatment of haemophilia, there may be provided anincreased offer of the factors VIII and/or IX. Moreover, factors may beadded which are conducive to the spreading-in and the growth offibroblasts and thus speed up the healing of the wound. For thispurpose, there may e.g. be provided a low content of fibronectin.

According to a preferred aspect of the invention at least one layer ofthe sheet material--preferably one or both thrombin-containing outerlayer(s)--contains fibroblast cells which are present in dry state andof which at least a fraction may be reactivated. Fibroblasts areelongated, spindle-shaped cells with long cell processes. They occur inthe loose connective tissue, where they form the collagenous as well aselastic fibers. For the present invention, fibroblasts of human originwhich have been reproduced by cell division in suitable nutrients comeinto consideration. For example, fibroblasts may be isolated fromjuvenile, mesenchymal tissue and slightly be reproduced by cell divisionin a basal medium according to "Eagle" of the company Bohringer,Mannheim, with an addition of bovine serum albumin. The fibroblast cellsare separated from the culture broth by centrifuging and washed with0.9% of NaCl solution. Thereupon, the fibroblast cells freshly obtainedin this case may be added to the selected glycoprotein solution and/orsuspension for producing a predetermined sheet-material layer.Alternatively, the freshly obtained fibroblast cells may be suspended ina protein-containing solvent and this suspension may be deep-frozenuntil further use.

The freshly obtained fibroblast cells or the fibroblast cells stored indeep-frozen state are added to the glycoprotein solution and/orsuspension used for the production of a predetermined sheet-materiallayer in such an amount that the finished dry sheet material contains10³ to 10¹⁰ fibroblast cells in dry state per 1 cm³ of the glycoproteinmatrix. An amount of approx. 10⁴ to 10⁶ fibroblast cells in dry stateper 1 cm³ of glycoprotein matrix of the finished dry sheet material isparticularly preferred.

It has been found within the scope of the present invention that atleast 10%, mostly 15% and more, of the fibroblast cells which afterdeep-freezing and lyophilization are present in dry state in the drysheet material may again be reactivated even after prolonged storage ofthe sheet material at room temperature when contacted with human serumand will spread into the wound area as well as into the substrateoffered with the sheet-material. Thus, when mention is made in thesepapers (specification and claims) of "fibroblast cells present in drystate", the term "fibroblast cells present in dry state" shall beunderstood to refer to and describe such peripheral conditions, e.g. inthe isolation, separation and, where applicable, storage of thefibroblast cells, in the preparation of the sheet-material layer and inthe finishing, sterilization and storage of the dry sheet material wherefrom the total number of the dry fibroblast cells presents at least 10%and preferably 15% or more may again be reactivated when contacted withhuman serum.

Besides, it has been found that also the presence of non-reactivatablefibroblast cells in the sheet material according to the invention mayhave a favourable influence, presumably through the increased offer ofcertain proteins, enzymes, active substances and factors.

The composition of the interstitial tissue is effected by polyanionicglycosamine glycans. These include the chondroitin sulphates A, B and C(chondroitin sulphate C=dermatane sulphate) and the keratane sulphate aswell as the sulphate-free hyaluronic acid. Glycosamine glycans arepolymeric carbohydrates (mean molecular weight mostly between 6,000 and12,000), which usually contain sulphate and acetate groups in addition.These glycosamine glycans bond proteins so that proteoglycans areformed, which, in their turn, may associate hyaluronic acid. Thus, theendogenous connective tissue is formed, into which the fibroblastsspread.

According to a further aspect of the invention there is provided in atleast one layer of the sheet material a content of these agents whichare specifically conducive to the growth of the fibroblasts and theirspreading-in. Especially chondroitin sulphate, the commerciallyavailable chondroitin sulphate mixture (chondroitin sulphate A and B),dermatane sulphate (chondroitin sulphate C), keratane sulphate andhyaluronic acid come into consideration as such agents. The use ofchondroitin sulphate is especially preferred. Preferably, the proportionof these agents conducive to the growth of the fibroblasts and theirspreading-in, especially of chondroitin sulphate, shall amount to 0.1 to1 mg per 1 cm³ of the glycoprotein matrix of the finished dry sheetmaterial. The specified agents, such as chondroitin sulphate, condroitinsulphate mixture, dermatane sulphate, keratane sulphate and hyaluronicacid are commercially available and may e.g. be obtained from thecompanies FLUKA, Fein-Chemikalien GmbH, Neu-Ulm, Federal Republic ofGermany, or SIGMA, Munich, Federal Republic of Germany.

A content of these agents conducive to the growth of fibroblasts andtheir spreading-in, especially of chondroitin sulphate, promotes to thegrowth and the spreading-in of the living fibroblasts carried along withthe serum in the wound area and therefore is appropriate also without anadditional offer of fibroblast cells present in dry state. However, thejoint presence of these agents conducive to the growth of thefibroblasts and their spreading-in, here especially chondroitinsulphate, and of dry-state fibroblast cells in the dry sheet material isparticularly preferred--and here preferably in at least one of thethrombin-containing outer layers.

Further, salts may be added, such as e.g. NaCl, CaCl₂, buffer salts,such as carbonate or the like, which remain in the matrix, activate thecoagulation factors and, where applicable, increase the solubility ofthe glycoproteins.

The contact layer, viz. the thrombin-containing layer, preferablycontains additionally adrenaline and/or ergotamine. Both substances arevasoactive, which has the consequence that the blood coagulates morequickly. Preferably, 0.05 to 0.1 mg of adrenaline per 1 cm³ ofglycoprotein matrix are provided; regarding the ergotamine there ispreferably provided an amount ranging from 0.5 to 10 μg per 1 cm³ of theglycoprotein matrix.

If antibiotics are provided, such as e.g. gentamycin (a known broadspectrum antibiotic), the same preferably shall also be contained in thethrombin-containing layer. The fibrinolysis inhibitors may be providedin all layers, but preferably are contained in the fibrinogen-containinglayer.

In the following the invention will be explained in detail by means ofpreferred embodiments with reference to the drawings, in which

FIG. 1 shows a double-layer embodiment of the sheet material; and FIG.2. shows a three-layered embodiment of the sheet material.

According to one embodiment, the sheet material according to theinvention is double-layered and consists of a thrombin-free layer, inthe glycoprotein matrix of which the fibrinogen is distributedsubstantially homogeneously, as well as of the fibrinogen-free layer, inthe glycoprotein matrix of which the thrombin is distributedsubstantially homogenously. The glycoprotein matrix of the respectivelayers may be made of the same or of different glycoproteins.

Such an embodiment is schematically shown in FIG. 1, where thethrombin-free layer is referenced 1 and the fibrinogen-free layer isreferenced 2.

With such a double-layer embodiment it is possible to conform thethrombin offer specifically to the wound area to be treated, forinstance, to provide for heavily bleeding wounds a high thrombin offerfor rapid haemostasis while the additional fibrinogen offer is small.

The thrombin-containing layer is intended for application onto thewound, and for this purpose it is suitably marked, for instance dyedwith haemoglobin.

Preferably, each layer 1 and 2 is of foam or fleece structure. Due toits large surface, this structure ensures good absorptive capacity. Theincreased contact area activates the coagulation factors contained inthe flow of blood as well as the factors additionally offered with thesheet material. A fleece formed of glycoprotein fibers has an evengreater mechanical stability than a corresponding foam structure; thismay be of importance, for instance, in the treatment of musclelacerations.

For each layer of the double-layer structure preferably a layerthickness of approx. 1 to 5 mm is provided. This range of the layerthickness may rapidly be soaked by the flow of blood so that thecoagulation factors offered with the sheet material are rapidlyactivated.

According to a further embodiment, the sheet material according to theinvention may be three-layered. Such an embodiment is schematicallyshown in FIG. 2, where the centrally disposed layer is referenced 4 andthe two outer layers are referenced 3 and 5.

In such a three-layered structure preferably the fibrinogen is containedin the centrally disposed layer 4 and the thrombin is contained in atleast one of the two outer layers 3 and 5. In some cases of application,for instance, in the case of sheet materials which are to be applied inthe abdominal cavity, e.g. for treating intestinal sutures, it may besuitable to cover the centrally disposed fibrinogen-containing layer onone side with a thrombin-containing layer and on the other side with alayer which is free both from thrombin and from fibrinogen but containscoagulation-inactive proteins, such as albumin or globulin. Thisprevents reliably that the resultant fibrin networks lead to an adhesionof the intestinal loops.

In a further embodiment of the three-layered sheet material thrombin iscontained in both outer layers. This embodiment is mainly suited for thetreatment of deeper muscle lesions or ruptures of soft organs (liver,spleen, pancreas), because the thrombin layer on either sides causesrapid closure of the numerous open capillaries in the mentioned organs.These thrombin-containing wound materials may be used advantageouslyalso in the case of more heavily bleeding surgical wounds, such as thosefrequently occurring in gynaecology or orthopaedics. Thethrombin-containing layers, which are intended for direct contact withthe wound, need not be marked particularly. When placed into the woundgap, both sheet material surfaces react with the adjacent wound surface.

In the three-layered structure, preferably a greater layer thickness isprovided for the central layer 4 than for the two outer layers 3 and 5.This permits forming the centrally disposed, fibrinogen-containing layer4 of such glycoproteins as are incorporated directly into the fibrinnetwork and, as a rule, only have a low mechanical strength, such ase.g. fibrin fission products. It is thereby possible to control themechanical stability of the wound closure under load. The mechanicalstrength may be further increased by the additional presence of collagenand/or modified collagens. The greater the offer of fibrinogen per unitof area of the central layer 4, the more fibrin will be formed, which,in its turn, increases the mechanical stability of the wound closureunder load.

According to a further embodiment of the invention, the single-stratumcentral layer 4 shown in FIG. 2 may be replaced by two or moreindividual layers, which results in a four-layered or even more-layeredstructure, wherein the composition of the inner layers may be matched topredetermined applications, for instance, a delayed release of activesubstance and accordingly an extended influence on the healing of thewound may be achieved. For example, it is possible to provide betweenthe outer contact layers a number of layers having differentactive-substance contents, which, when dissolving, release thesubstances contained therein into the wound area in a graduated orderrelative to time. Such an embodiment is recommendable, for instance, forthe treatment of infected bone fractures, to which antibiotics have tobe supplied for a prolonged period of time, which then are released in agraduated order relative to time due to the different decompositionrates of the layers.

Regarding the optimum composition of the sheet material according to theinvention, especially the following aspects are applicable:

The dissolution rate of the glycoprotein support material determines thefield of use of the fibrinogen fleece. Where a long residence time ofthe wound closure material is desired, collagen must be used as asupport material. Where only a rapid closure of the wound is to beachieved, but the material is to be decomposed rapidly by endogenousproteases, recourse must be taken to other plasma proteins (albumin,globulin, fibrin).

Therefore, an optimum composition shall start with thrombin-containingouter layers.

For this purpose, albumin or globulin are suited as support materials(in dry form).

Small additions of collagen increase the mechanical stability in thislayer.

An additional content of fibroblast cells present in dry state and/or ofagents conducive to the growth of the fibroblasts and theirspreading-in, and here especially chondroitin sulphate, in at least oneof the thrombin-containing outer layers promotes the healing of thewound.

The fibrinogen-containing layer shall be disposed centrally, whereas thefollowing outer side must not comprise anything but thrombin orcovering, coagulation-inactive proteins such as albumin or globulin.

In a particularly preferred embodiment of the sheet material, the outerlayer consists of a thrombin-containing layer (made of an aqueous 5%albumin solution containing 200 to 1,000 units of thrombin per 1 ml)having a thickness of approx. 2 mm. The following collagen supportmaterial contains the fibrinogen. This layer is obtained bydeep-freezing from an aqueous, saline collagen solution with 1 mgfibrinogen per ml of solution. The collagen content, which mostly iswithin the range from 0.8% to 2.0%, may be lowered to a finalconcentration of 0.25% provided a fibrin suspension or another plasmaprotein is additionally added as a stabilizer. The second outer layer isformed by albumin from a 5% aqueous solution. When required, thrombin isadded also to this outer layer.

The invention is further explained by the following examples, which,however, do not constitute a limitation thereof.

EXAMPLE 1

To a commercially available (company Pentapharm, Basel, Switzerland)collagen solution containing 1% of collagen in saline water 0.5 mg ofsolid, microcrystalline fibrinogen (obtained according to theabove-described process) as well as 10 mg of solid albumin are added,per 1 ml of solution respectively. Agitation is carried out at roomtemperature for several minutes until fibrinogen and albumin havedissolved entirely.

This solution is poured into a flat inert dish having a plane bottomuntil a liquid layer having a thickness of 3 mm is obtained. Thereupon,deep-freezing takes place; for this purpose, the dish including itscontent is kept at a temperature of -40° C. for approx. 45 min.

Thereupon, so much thrombin-containing collagen solution is poured ontothe surface of the resultant ice layer that again a liquid/ice layerhaving a thickness of approx. 2 mm is formed. For pouring, this secondsolution suitably is at room temperature. Thus, the previously formedice layer melts on the surface, and after the renewed deep-freezing,followed by a lyophilization, a very stable bond between the layers isobtained without occurrence of an appreciable reaction of the fibrinogendue to the low temperatures and the short reaction period. For formingthis second solution, thrombin ("Topostasin" of Hoffmann LaRoche,Grenzach, Baden) was dissolved in the above-mentioned (however,fibrinogen-free) collagen solution (200 units of thrombin per 1 ml ofsolution). Deep-freezing is carried out again; for this purpose atemperature of -40° C. is maintained for approx. 45 min. Thereupon,lyophilization is carried out under the usual conditions.

After termination of the drying operation, the sheet material iscarefully loosened from one edge of the mold bottom and thereupon pulledoff in the form of a shred of material. A large-area, supple, flexibleprotein fleece having a layer thickness of approx. 5 mm is obtained. Theshred of material is deposited on an Al foil for a short time, cut tothe desired dimensions, the resultant pieces are put into a recessedmold of synthetic material and the latter is closed with an Al foil.Thereupon, the package including its content is sterilized with X-rays(dose: 3,000 rad for 3 min.).

At room temperature, the resultant product has a substantially infinitestorage stability without any appreciable loss of activity. Forapplication, the package is opened and the double-layer sheet materialis placed with slight pressure onto the bleeding wound with thethrombin-containing layer in direct contact with the same. The sheetmaterial rapidly resorbs the blood and/or plasma issuing from the woundand very rapidly causes the same to coagulate--depending on the escapeof blood in the wound area, at any rate, within 3 to 5 min., wherein thesheet material turns red and forms a stable fibrin/collagen cake on thewound area.

EXAMPLE 2

A further, double-layer sheet material is produced in a waysubstantially analogous to that of Example 1. In contrast to Example 1,an aqueous, saline (0.9% of NaCl, 0.025 molar of CaCl₂) 5% albuminsolution, to which 100 units of thrombin (Topostasin), 50 units ofprothrombin (PPSB preparation of the company Immero AG, Vienna), 1,000units of gentamycin as an antibiotic are added per 1 ml, is used to formthe thrombin-containing layer. Furthermore, some crystals of haemoglobinare added to the solution as a whole so as to mark the contact layer.This solution is poured up to a layer depth of 2 mm into the mold and issubjected to deep-freezing (-40° C.). A 1% aqueous collagen solutioncomprising 3 mg of fibrinogen per 1 ml is poured up to a layer depth of3 mm onto the resultant ice layer, deep-freezing is applied again,whereupon lyophilization is carried out.

EXAMPLE 3

The production of a three-layered sheet material takes place in a waywhich is substantially analogous to that of Example 1. 50 units ofthrombin are added to an aqueous 3% albumin/0.5% collagen solution, per1 ml thereof. The solution is poured up to a layer depth of 2 mm into amold having an inert surface and subjected to deep-freezing (-40° C.). A1% collagen solution comprising 3 mg of fibrinogen and 2 mg of aprotinin(as a fibrinolysis inhibitor) per 1 ml of solution is poured up to alayer depth of 4 mm onto the frozen layer and deep-freezing is alsoapplied. Finally, the above albumin/collagen/thrombin solution is againapplied up to a layer depth of 4 mm, and after deep-freezing the mold,which is provided with three layers, is lyophilized. The resultant drysheet material is lifted at one side under exclusion of moisture,loosened from the mold by means of a blunt spatula and after cutting tothe specified size sealed in the packaging foil and the packageincluding its content is sterilized.

EXAMPLE 4

Thrombin is introduced into a 5% human globulin solution (1,000 units ofthrombin per 1 ml of solution) and this solution is subsequently foamedwith nitrogen gas.

The foamy material is poured into the lyophilization mold and subjectedto deep-freezing. The height of the layer is approx. 3 mm.

A fibrinogen-containing collagen solution (10 mg of fibrinogen and 0.075mg of adrenaline (commercially available preparation) in 1 ml of 1%collagen solution respectively) is applied without foaming and up to alayer height of 5 mm onto the thrombin-containing layer and alsosubjected to deep-freezing. The final layer is formed from the abovethrombin-containing 5% human globulin solution, which is applied as afoam up to a total height of 8 mm. After lyophilization, the material iscut to shape and packaged. After packaging, sterilization by radiationis carried out.

EXAMPLE 5

Non-linked fibrin is pulverized and suspended in an amount of 0.6 g per1 ml of water. 1% of human albumin is added as an admixture. Thrombin isadded in an amount of 300 units per 1 ml. The fibrin-albumin-thrombinsuspension is poured up to a level of 2 mm into a mold and immediatelysubjected to deep-freezing. A 1% collagen solution comprising 1 mg offibrinogen per 1 ml and 1 mg of fibrin particles per 1 ml is poured upto a layer depth of 4 mm onto the ice layer. Finally, the abovethrombin-containing albumin-fibrin suspension is applied onto thedeep-frozen second layer, subjected to deep-freezing and themulti-layered frozen material is lyophilized.

The removable dry protein substance forms a stable sheet material, whichmay be cut easily.

When used as a material which is applied on wounds, it may easily bemanipulated, may be bent, which is necessary especially in the treatmentof intestinal sutures, and absorbs blood or plasma in the wound area.

EXAMPLE 6

The following solutions/suspensions are used to produce a four-layeredsheet material--substantially in a way which is analogous to that ofExample 1:

(a) 50 units of thrombin per 1 ml of 5% aqueous, saline albuminsolution;

(b) 50 units of prothrombin per 1 ml of a 2.5% aqueous albumin solution;

(c) 2 mg of fibrinogen, 5,000 units of gentamycin, 8 μg of ergotamine,10,000 units of α₂ -antiplasmin, 10,000 units of α₂ -macroglobulin per 1ml of 5% aqueous albumin solution;

(d) 2.0% aqueous collagen solution with addition of 5,000 units ofgentamycin per 1 ml of solution.

For producing the sheet material, the solution "a" is poured up to alayer depth of 2 mm into a mold having an inert surface and subjected todeep-freezing. The solution "b" is poured thereon up to a layer depth of2 mm, the solution "c" is poured thereon up to a layer depth of 2 mm andfinally the solution "d" is poured thereon up to a layer depth of 4 mm,after deep-freezing of the respective preceding layer. The finishedthawed dry preparation has a compressible fleece structure with asubstantially stable area and a total layer thickness of approx. 10 mm.The formation of multiple layers permits the introduction of variousreactants or drugs so that, when infectious wound areas are treated, theantibiotics are released with delay and a longer protection againstinfections is ensured. Collagen in the soluble form, as used in thepreparation of this product, is identifiable in the healing wound areafor more than 14 days. Within this period of time, the drugs offered incombination with the sheet material diffuse into the surrounding tissue.The diffusion rate is determined by the association e.g. of theantibiotic with the collagen support material or fibrin.

EXAMPLE 7

Substantially Example 1 is repeated; however, additionally fibroblastcells are added to the thrombin-containing collagen solution.

Fibroblast cells originating from juvenile, mesenchymal human tissuewere cultured in basal medium according to "Eagle" of the companyBohringer, Mannheim, Federal Republic of Germany, to which additionallybovine serum albumin had been added. These fibroblast cells wereseparated from their nutrient medium by centrifuging, washed with 0.9%of NaCl solution and thereupon taken up in a 5% human albumin solutionand suspended without destruction of the cells.

Such a proportion of this fibroblast suspension is added to the solutionused in Example 1 and obtained by dissolution of "Topostasin" in asaline 1% collagen solution that the thrombin-containing collagensolution contains 10³ to 10⁶ fibroblast cells per 1 ml of collagensolution. Finally, this collagen solution containing thrombin andfibroblasts is poured onto the already form ice layer offibrinogen-containing collagen solution and, without foaming, subjectedto deep-freezing. Subsequently, both layers are jointly lyophilized,cut, packaged and sterilized. In order not to harm the fibroblast cells,the sterilization is carried out at a lower dose for a prolonged periodof time, e.g. 1,000 rad for 9 min.

EXAMPLE 8

Substantially Example 7 is repeated; however, additionally chondroitinsulphate is added to the solution containing thrombin and fibroblasts.

In particular, a saline 1% collagen solution is so enriched withfibroblast cells obtained by centrifuging from their culture broth that1 ml of solution contains approx. 10³ fibroblast cells. Thereupon,commercially available chondroitin sulphate mixture (chondroitin A andB, mean molecular weight approx. 8,000, obtained from the company FLUKAFeinchemikalien GmbH, Neu-Ulm, Federal Republic of Germany) is added inan amount of 1 mg per 1 ml of solution and dissolved. Thereupon, 30units of thrombin per 1 ml of solution are added.

This solution is applied with a layer thickness of 3 mm onto a preformedice layer of fibrinogen-containing collagen solution (3 mg of humanfibrinogen per 1 ml of saline 1% collagen solution and deep-frozen.Subsequently, both layers are jointly lyophilized, cut, packaged andsterilized.

EXAMPLE 9

A three-layered sheet material is produced in a way substantiallyanalogous to that of Example 7.

Fibroblast cells are separated by centrifuging from their culture brothand suspending in a saline 1% albumin solution. After adjustment of aconcentration of 10⁵ fibroblast cells per 1 ml of solution, 0.3 mg ofchondroitin sulphate and 300 units of thrombin--each per 1 ml ofsolution--are added. The resultant first suspension is poured up to alayer thickness of 4 mm into a flat 3.5×8 cm mold and deep-frozentherein.

For preparing the central layer, fibrinogen (5 mg per 1 ml of solution)is taken up in saline, 1% collagen solution. This second solution ispoured onto the ice layer of the first suspension and also subjected todeep-freezing.

For producing the other outer layer, the above-mentioned firstfibroblast suspension is again used in an albumin solution containingthrombin and chondroitin sulphate.

After renewed deep-freezing and the joint lyophilization, athree-layered sheet material having a total layer thickness of 12 mm isobtained.

What is claimed is:
 1. A method of making a resorptive sheet materialfor closing and healing wounds, consisting essentially of a glycoproteinmatrix containing substances which cause and, respectively, areconducive to the coagulation of blood, including fibrinogen andthrombin, wherein the sheet material is dry and of multi-layeredstructure; at least one layer is free from thrombin and contains in theglycoprotein matrix thereof, the fibrinogen in a substantiallyhomogeneously distributed form; and at least one further layer is freefrom fibrinogen and contains in the glycoprotein matrix thereof thethrombin in a substantially homogeneously distributed form; whichprocess comprises adding fibrinogen to a thrombin-free, predominantlyaqueous solution or suspension of the glycoprotein and processing saidsolution or suspension to form a sheet material layer; and applying ontothe full surface of this layer at least one further layer which, in itsturn, has been obtained from a further layer, fibrinogen-free andthrombin-containing solution or suspension of the same or of anotherglycoprotein.
 2. A method as claimed in claim 1 which additionallyincludes enriching the thrombin-containing solution or suspension of theglycoprotein with active fibroblast cells.
 3. A method as claimed inclaim 1 which comprises additionally enriching the thrombin-containingsolution or suspension of the glycoprotein with an agent conducive tothe growth or spreading-in of fibroblasts, or both.
 4. A method asclaimed in claim 1 which comprises additionally adding chondroitinsulphate to the thrombin-containing solution or suspension of theglycoprotein.
 5. A method as claimed in claim 1 wherein for producingthe sheet material layer the respective solution or suspension, or both,is applied onto an inert surface, where it is subjected to deep-freezingand, finally, to lyophilization.
 6. A method as claimed in claim 1 whichcomprises producing the layers independently of one another; moisteningat least one layer surface with water; and placing a further layer ofdifferent composition onto the moistened layer surface.
 7. A method asclaim 1 which comprises applying a solution or suspension, or both, ofone composition onto an inert surface, where it is subjected todeep-freezing; and applying onto the resultant ice-layer surface, whenstill in the deep-frozen state, a solution or suspension, or both, ofdifferent composition and subjecting it to deep-freezing; and jointlysubjecting all layers to lyophilization.
 8. A method as claimed in claim7 which comprises foaming before one or each of the deep-freezingoperations.